Structural basis for regulation of the nucleo-cytoplasmic distribution of Bag6 by TRC35 Jee-Young Mocka, Yue Xub, Yihong Yeb, and William M. Clemons Jr.a,1 aDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; and bLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD 75534 Edited by Jeffrey L. Brodsky, University of Pittsburgh, Pittsburgh, PA, and accepted by Editorial Board Member F. Ulrich Hartl, September 20, 2017 (received for review February 23, 2017) The metazoan protein BCL2-associated athanogene cochaperone 6 heterotetrameric Get4–Get5 complex (3, 13), but the addition of (Bag6) forms a hetero-trimeric complex with ubiquitin-like 4A and Bag6 leads to different interactions. transmembrane domain recognition complex 35 (TRC35). This Crystal structures of fungal Get4 revealed 14 α-helices that can Bag6 complex is involved in tail-anchored protein targeting and be divided into N-terminal domains (NTD) and C-terminal do- various protein quality-control pathways in the cytosol as well as mains (CTD) (Fig. 1A and Fig. S1A)(14–16). The NTD contains regulating transcription and histone methylation in the nucleus. conserved residues (Fig. S1C) involved in binding and regulating Here we present a crystal structure of Bag6 and its cytoplasmic Get3 (13, 15), while the CTD forms a stable interaction with the retention factor TRC35, revealing that TRC35 is remarkably Get5 N terminus (Get5-N) (15). The Get5 interface includes a conserved throughout the opisthokont lineage except at the hydrophobic cleft in Get4 formed by a β-hairpin between α13 and C-terminal Bag6-binding groove, which evolved to accommodate α14 that binds the helix in Get5-N (15). Sequence alignment of Bag6, a unique metazoan factor. While TRC35 and its fungal ho- Get4/TRC35 and Get5/Ubl4A led to predictions that the Get4 molog, guided entry of tail-anchored protein 4 (Get4), utilize a β-hairpin and Get5 N-terminal extension are absent in metazoans conserved hydrophobic patch to bind their respective partners, (Fig. 1 A and B) (15), which suggested a different structural or- Bag6 wraps around TRC35 on the opposite face relative to the ganization for the metazoan Bag6–TRC35–Ubl4A complex. Get4–5 interface. We further demonstrate that TRC35 binding is In metazoans, Bag6 abrogates the interaction of TRC35 with critical not only for occluding the Bag6 nuclear localization se- Ubl4A by separately binding these factors (3, 17) resulting in a BIOCHEMISTRY quence from karyopherin α to retain Bag6 in the cytosol but also heterotrimer. The interface between TRC35 and Bag6 had pre- for preventing TRC35 from succumbing to RNF126-mediated ubiq- viously been mapped to the CTD of TRC35 and the region on uitylation and degradation. The results provide a mechanism for Bag6 that contains the bipartite nuclear localization sequence regulation of Bag6 nuclear localization and the functional integrity (NLS) (3, 18). Overexpression of TRC35 results in Bag6 retention of the Bag6 complex in the cytosol. in the cytosol (18), suggesting that TRC35 plays a role in de- termining the nucleo-cytoplasmic distribution of Bag6. Potential tail-anchor targeting | X-ray crystallography | tail-anchor recognition roles for Bag6 in the nucleus are varied and include modulation of complex | proteasome-dependent degradation | GET pathway p300 acetylation (19, 20), facilitation of histone methylation (21), and regulation of DNA damage signaling-mediated cell death ail-anchored (TA) membrane proteins contain a single, (22). How Bag6 localization is regulated remains unclear. TC-terminal, hydrophobic transmembrane-helix domain (TMD) In this study, we present a crystal structure of a complex that (1). The TMD, which acts as the endoplasmic reticulum (ER) includes TRC35 from Homo sapiens (Hs) and the TRC35- targeting signal, emerges from the ribosome after translation binding domain of HsBag6. The structure of TRC35 reveals termination necessitating posttranslational targeting and in- sertion. Transmembrane domain recognition complex (TRC) Significance proteins in mammals and guided entry of tail-anchored proteins (GET) in fungi are the best-characterized molecular players in The metazoan protein BCL-2–associated athanogene cochaper- TA targeting (2). In the TRC pathway, TA proteins are captured one 6 (Bag6) acts as a central hub for several essential cellular by the small glutamine-rich tetratricopeptide-repeat protein A processes, including immunoregulation, gene regulation, apo- (SGTA) after release from the ribosome then transferred to the ptosis, and proteostasis. These roles are in both the nucleus and ATPase TRC40, a process facilitated by the hetero-trimeric Bag6– the cytosol, but the mechanism by which Bag6 traffics between TRC35–Ubl4A complex (3, 4). TA binding stimulates hydrolysis of these compartments remains elusive. Here we present the crystal ATP by TRC40 (5) leading to release of the TRC40–TA complex structure of Bag6 in complex with its cytoplasmic retention factor that then localizes to the ER membrane via its interaction with transmembrane domain recognition complex 35 (TRC35) and suggest a mechanism of regulation for the nucleo-cytoplasmic calcium-modulating ligand (CAML) (6). Release of the TA sub- transport of Bag6. strate for insertion is facilitated by CAML and tryptophan rich basic protein (WRB) (7). Notably, loss of WRB in mouse cardiomyocytes Author contributions: J.-Y.M., Y.Y., and W.M.C. designed research; J.-Y.M., Y.X., and Y.Y. and hepatocytes results in partial mislocalization of TA proteins (8), performed research; J.-Y.M., Y.X., and Y.Y. contributed new reagents/analytic tools; suggesting that other pathways, including the signal-recognition J.-Y.M., Y.Y., and W.M.C. analyzed data; and J.-Y.M. and W.M.C. wrote the paper. particle (SRP) (9), SRP-independent targeting (SND) proteins The authors declare no conflict of interest. (10), the Hsc70 family of chaperones (11), and ubiquilins (12), can This article is a PNAS Direct Submission. J.L.B. is a guest editor invited by the Editorial compensate to target TA in vivo. Board. Structural information for the mammalian proteins is sparse; Published under the PNAS license. however, homology to the equivalent fungal counterparts, Get1– Data deposition: The atomic coordinates and structure factors reported in this paper have been deposited in the Protein Data Bank (PDB), https://www.wwpdb.org (PDB ID code 5 and Sgt2, allow for parallels. Bag6 is unique among the 6AU8). mammalian proteins, and its introduction alters the molecular 1To whom correspondence should be addressed. Email: [email protected]. architecture of the central hand-off complex. For TA targeting, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the heterotrimeric Bag6 complex is equivalent to the fungal 1073/pnas.1702940114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1702940114 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 A α11 α12 Get5-N Get4 Hsap KSSASVVFTTYTQKHPSIEDGPPFVE-----------------PLLNFIWFLLLAVDG 249 Aque PDEFGQLLVDLSYKEAKPHEIEMFIT-------------------QAVLQLLVLEKKD 229 Spom LISAWNSLETFTKHFTKSNAPDVENMSFDGK----DFPVFKEYPQMNFLHLLIFTAYR 239 Ncas IRFVNQAKEAFLTSFINKYAPKVEITEKNLGSGVFKMYYFESYKSLNFLQLLVLTCQT 260 Scer ISFAHESKDIFLERFIEKFHPKYEKIDKNG----YEIVFFEDYSDLNFLQLLLITCQT 257 Afum LRNANKAFLVFTSRLSSSNTSLGVQEVSSAS---SDVRVFPSLPLLNFISMLLLTIQR 278 Fig. 1. The structure of the Bag6NLS–TRC35 hetero- Ncra VRAANTSYRAFVSALTAENSSLGVQNVESSQG--SDIRIFPSLPLLNLLGLLLLAVQK 256 Smus TRAANKALLLFTSRLSTSHPGLGVQSISSPS---SDIRIYPSLPLLNFLGLLLLAVQR 246 dimer. (A) The structure of Saccharomyces cerevisiae – B C N Get4 Get5N complex [Protein Data Bank (PDB) ID code: 16 3LKU], Get4 in rainbow and Get5 in magenta. Sequence alignment of TRC35/Get4 from Homo sapiens, Amphi- 15 N 6 medon queenslandica, Schizosaccharomyces pombe, C 13 14 4 2 N 8 Naumovozyma castelli, Saccharomyces cerevisiae, As- pergillus fumigatus, Neurospora crassa,andSphaerulina musiva. The secondary structure based on fungal Get4s 90° 12 10 5 3 1 9 7 is highlighted above the text. (B, Left) The overall structure of Bag6–TRC35 heterodimer in cylinder rep- C Bag6 TRC35 11 resentation with Bag6 in light pink and TRC35 in rain- NLS bow. The nuclear localization sequence is highlighted in α12 90° V254 ‘ ’ CDα13 α13 V1008 sticks on Bag6. (B, Right) A 90° in plane rotated bottom α12 M271 view. The 16 helices of TRC35 are labeled from N to C W1012 V257 M1040 terminus. (C) Comparison of the C-terminal faces of α11 F188 Y1036 TRC35 and Get4 that bind Bag6 and Get5, respectively. W1004 F242 L258 The arrows highlight the significant structural differ- I1016 L1032 ence in the residues between α11 and α12. (D) Zoomed α11 Y262 F195 in view of the regions, defined as interface I and II. – Interface I Interface II Hydrophobic residues that are involved in Bag6 Get5Get4 Bag6 TRC35 TRC35 dimerization are shown as sticks. the conserved architecture relative to fungal Get4 homologs. are well conserved (Fig. S1 C and D). As predicted from se- Surprisingly, Bag6 utilizes the same conserved pockets on TRC35 quence alignment, the β-hairpin in Get4 is replaced by a shorter that are recognized by Get5 on Get4; yet the extended domain loop in TRC35 (Fig. 1C, arrows). In fact, sequence alignment of wraps around the opposite face of the α-solenoid. The resulting the predicted Get4 proteins of selected opisthokonts revealed interaction leads to masking